The present invention relates to a group of compounds and a method for inhibiting certain enzyme systems that mediate a variety of physiological responses in mammals. More particularly, the present invention relates to a compound and method for inhibition of phospholipase A2, and cyclooxygenase-2, which are enzymes that catalyze a cascade of biochemical reactions that lead to the mediation of pain, fever, inflammation and other functions.
Human beings and other mammals spend energy continuously defending against a vast array of invasive pathogenic organisms including bacteria, viruses, fungi and other intracellular and extra-cellular parasites in addition to other potentially harmful agents that are capable of upsetting homeostasis. In response, humans and other mammals maintain many mechanisms capable of processing and defending against such antigens and agents. The biological response to attack and injury is mediated through the formation of a series of structurally related compounds called eicosanoids, which include the prostaglandins, the leukotrienes, and the thromboxanes. Master enzymes known as phospholipase A2 and phospholipase C regulate the formation of these highly potent compounds.
Phospholipase A2 is a heat-stable, calcium dependent enzyme that catalyses the hydrolysis of the 2-acyl bond of 3-n-phosphoglycerides. It has a molecular weight of about 30,000 Daltons. Phospholipase A2 has been found in many human tissues including platelets, chondrocytes, placenta, cartilage, peritoneal calls and peritoneal fluid and spleen. (Vades, p., Puzanski, W., Soluble phospholipase A2 in human pathology: clinical-laboratory interface. Biochemistry, molecular biology, and physiology of phospholipase A2 and its regulatory factors. Ed AB Mukherjee, Plenum Press, New York, 1990.) High levels of phospholipase A2 are found in synovial tissue and it has been shown that activity of rheumatoid arthritis significantly correlated to the levels of serum phospholipase A2. (Vadas, P., Pruzanski, W. and Stefanski, E., Characterization of extracellular phospholipaseA2 in human synovial fluids. Life Sci. 36: 579, 1985.)
Substantial evidence has been found (above reference) that excessive concentrations of extra-cellular phospholipase A2 may initiate and propagate inflammation and cause cellular damage. In addition phospholipase A2 was also found to modulate various aspects of phagocytic activity, vascular tone and permeability. A strong correlation between phospholipase A2 activity and certain human diseases have also been identified. A few such diseases are listed in table 1 below.
FIG. 1 illustrates the key role that phospholipase A2 and cyclooxygenase-2 are currently understood to play in the formation of potent, biologically active substances that mediate a variety of conditions and disease states. The process of the formation of prostaglandin""s, leukotrienes, lipoxins, and thromboxanes in addition to lysosomal enzyme release, bactericidal activity, pro-inflammatory eicosanoids, PAF and lysophosphatides and reactive oxygen species, begins on the surface of specialized cells including osteoblasts, endothelial cells, chondrocytes, synoviocytes, and renal mesangial cells. The major constituents of these, and all other, cell membranes are phospholipids. The biochemical conversion of these important molecules to arachidonic acid is catalyzed by phospholipase A2. Arachidonic acid is further converted to leukotrienes, lipoxins, thromboxanes and prostaglandins. The latter two species are formed by way of chemical conversion catalyzed by two other important enzymes known as cyclo-oxygnease-1 and cyclooxygenase-2. The arachidonic acid cascade is a well know pathway leading to the mediation of pyrogenicity, vasoconstriction, increased vascular permeability, contraction of smooth muscle, inflammation, and pain.
Found in every cell of the human body, albeit to varying degrees, prostaglandins have profound physiologic effects including mediation of pain and inflammation. The prostaglandins (PG) are a family of lipid-soluble hormone-like molecules produced by different cell types in the body. For example, macrophages and monocytes are large producers of both PGE2 and PGF2, neutrophils produce moderate amounts of PGE2, and masts cells produce PGD2. It is important to note that, unlike histamine, prostaglandins do not exist free in tissues vacuoles, but have to be synthesized and released in response to an appropriate stimulus. This synthesis is dependent on phospholipase A2 and cyclooxygenase-2.
Thromboxanes are produced by monocytes and macrophages, as well as by platelets. Thromboxanes are involved in causing platelets to aggregate and constrict blood vessels and airways. These effects are some what opposed by the action of prostacyclin (PGI2), which is a potent vasodilator.
Leukotrienes (LT) exist in a number of varieties, and cause the chemotaxis (directed locomotion) and/or chemokinesis (general cell movement) of a number of cell types including neutrophils. The synthesis of LTB4 is inhibited by colchicines, an anti-flammatory agent used for treatment of gout. The mixture of LTC4, LTD4 and LTE4 originally called slow reacting substance of anaphylaxis is produced by a wide variety of smooth muscle, mainly in the bronchus, and have effects on mucous secretions. Inhibition of the formation of these substances is a useful therapeutic modality in asthma.
Lipoxins (LX) are a family of molecules that are thought to stimulate changes in microcirculation. For example, LXA4 induces rapid arteriolar dilation and can also antagonize LTD4-induced vasoconstriction. This suggests that LXA4 may regulate the action of vasoconstrictor leukotrienes. LXA4 can block neutrophil chemotaxis induced both LTB4 and N-formyl-oligopeptieds. Both LXA4 and LXB4 inhibit cytoxicity of natural killer T cells and thus there inhibition can be useful in allowing the immune system to remain intact while fighting infections and cancer.
Inflammation is a complex response of the body in response to damage of its cells and vascular tissues. The elucidation of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response.
Basic symptoms of inflammation include redness, swelling, heat, pain, and deranged function. These signs and symptoms are thought to be due to extravasation of plasma and infiltration of leukocytes into the site of inflammation, as a result of cell damage. Early investigators considered inflammation a primary host defense system. Indeed inflammation is the key reaction of the immune response, but in fact, inflammation is more than this, since it can lead to death, as anaphylactic shock, or debilitating diseases, as in arthritis or gout.
What is needed is a novel lipid compound that is capable of reducing inflammation and certain related disease conditions, while avoiding disadvantages of the prior art, preferably without unacceptable side effects.
The present invention comprises a group of related lipid molecules, and methods for using the group of related lipid molecules, for inhibiting certain enzyme systems including phospholipase A2 and cyclooxygenase-2, for the mediation of pain, fever, inflammation and other functions including phospholipase A2 and cyclooxygenase-2. The ability of the group of related lipids of the invention to inhibit enzyme systems including phospholipase A2 and cyclooxygenase-2 was surprising and unexpected. The group of related lipid molecules of the invention may be characterized by specific structural characteristics, or by specific biological activity, including inhibition of the previously listed enzymes. The group of related lipid molecules of the invention may be synthesized using known chemical methodologies, or isolated and purified from biological materials using known methods for isolating desired lipids. The group of related lipid molecules of the invention may be altered by known chemical means to produce various derivatives or analogues. This invention also relates to forms of the lipids of the invention which, upon administration to a patient, undergo biotransformation into an active form.
With regard to the structural characteristics, the group of related lipid molecules of the invention share and may preferably be characterized by the following three structural features: a backbone or linking structure, a polyethyleneglycol (PEG) chain, and a tail group having two long chain fatty acids. The long chain fatty acids preferably range between 11 and 25 (inclusive) carbons in length, and may be saturated, unsaturated, or polyunsaturated. Each long chain fatty acid is preferably of the same length, and saturation, but in some embodiments the chains may differ. The PEG groups may be various lengths, but is preferably between 100 and 5,000 daltons, and more preferably between 500 and 2,000 daltons. While a glycerol backbone or linking structure is preferred, in alternate embodiments, backbone structures including only two carbon, or more than three carbons may be useable. Furthermore, related compounds only a single long chain fatty acid or three long chain fatty acid s, instead of two long chain fatty acids, may also be effective in inhibiting phospholipase A2 and cyclooxygenase-2, and are considered part of the current invention.
In a preferred embodiment, the lipid molecules of the invention, and compounds and mixtures thereof, may be administered to an animal or a humans as a pharmaceutical composition, a foodstuff, a food additive, a dietary supplement, or a cosmetic. The composition of the invention may be administered selectively or systemically, and delivery methods include but are not limited to oral and topical. The compound of the invention may be particularly suited for delivery as a component of a liposome or micelle. Useable systemic delivery methods may include but are not limited to use of the compound of the invention in -the form of a tablet, a capsule, a syrup, a dragee, a suspension, an elixer, a solution, a powder, granules, an emulsion, microspheres, nanospheres, lipid vesicles, polymeric vesicles, or an injectable. Useable topical delivery methods may include but are not limited to use of the compound of the invention in the form of an ointment, a cream, a milk, an impregnated pad, a gel, a spray, and a lotion.
The invention also includes methods for administering the lipids of the invention in effective amounts through a variety of know means including as a pharmaceutical, foodstuff, dietary supplement, or cosmetic.
Inflammation related diseases that may be treated include but are not limited to rheumatoid arthritis, osteoarthritis, psoriasis, monoarthritis, gout, collagen vascular disease, pancreatitis, peritonitis, sepsis and shock, renal failure.